PROJECT SUMMARY Soluble adenylyl cyclases generate a cAMP signal that breaks down microtubules leading to endothelial cell barrier disruption. The P. aeruginosa type 3 secretion system effector is one such soluble cyclase. However, we have discovered that this enzyme is a promiscuous nucleotidyl cyclase capable of generating both purine and pyrimidine (i.e. non-canonical) cyclic nucleotide monophosphates, including cGMP, cUMP, and cAMP. The cAMP signal, and to a lesser extent the cGMP signal, activates protein kinase A, which phosphorylates tau leading to microtubule breakdown. Phosphorylated tau accumulates inside the endothelium for several hours as a high molecular weight oligomeric form, and is then released from the cell into the supernatant in vitro, and bronchoalveolar lavage and blood in vivo. Preliminary data suggest cellular release of high molecular weight tau is stimulated by cUMP. Our previously published work and recent preliminary studies indicate high molecular weight tau retrieved from cellular supernatant fractions is heat stable, protease resistant, insoluble in certain detergents, insensitive to RNase and DNase treatments, and can be resolved in a 30-50% ammonium sulfate fraction by column chromatography. Further, high molecular weight tau is transmissible between cells, leading to inter-endothelial cell gap formation, increased permeability and cytotoxicity; pulmonary microvascular endothelial cells are especially sensitive to this injury. We have searched for a means to prevent the transmissible cytotoxicity as an anti-inflammatory therapy. We have recently discovered that prion protein is expressed in lung endothelium. Whereas prion antibody treatment initiates a pro-survival signal and prevents tau-induced hyperpermeability and cytotoxicity, prion protein genetic deletion increases endothelial sensitivity to the high molecular weight tau. Hence, this proposal tests the hypothesis that ExoY generates purine and pyrimidine cyclic nucleotides in endothelium and induces release of high molecular weight tau capable of generating transmissible hyperpermeability and cytotoxicity, an effect prevented by antibody ligation of prion protein.